Lyme disease was once restricted to coastal North Atlantic and selected Upper Midwest communities, but the distribution and prevalence of this zoonosis has greatly expanded in the last two decades. The aggressively human biting deer tick vector of the agent of Lyme disease was first recognized to maintain a distinct lineage of Powassan virus (POW) in 1997, but human encephalitis cases attributable to ?deer tick virus? was recognized only in 2006. Severe neurologic disease, a hallmark of classical Powassan fever, remained rare in residents of Lyme-endemic sites until recently. Encephalitis cases attributed to Powassan virus are now being increasingly reported from New England and the upper Midwest. The biological basis for the recent zoonotic emergence of POW requires analysis. The Eurasian tick borne encephalitis virus complex (TBEV) comprises diverse species (including POW), subtypes and geographic isolates that vary in their capacity to cause human disease, and there is a rich literature concerning variation in the genetic, phenotypic and clinical characteristics of Eurasian TBEV that should guide our analysis of the potential public health burden of POW in North America. Our overaraching hypothesis is that Powassan virus, like TBEV, also comprises lineages, genotypes, or populations that differ in capacity to cause human disease. We propose to test this hypothesis using our existing geographic isolates of POW as well as additional isolates that we shall collect for their capacity to cause neurologic disease using a published mouse model for TBEV neurotropism. In addition, it may be that certain lineages, genotypes, or populations of POW are more efficiently transmitted (particularly viral dose that is transmitted) by the human biting deer tick vector of Lyme disease, and this helps to explain the emerging epidemiological situation. Accordingly, we shall also determine whether geographic isolates may differ in their capacity to infect and be transmitted by ticks. Finally, we shall apply the powerful tools of whole genome sequencing to analyze the population structure of Powassan virus as well as identify any genetic correlates of neurotropic capacity. Taken together, these observations may help explain the changing epidemiology of an arbovirus that has been silently enzootic for over 2 decades in Lyme disease endemic sites, but is now apparently an emerging zoonosis. Ultimately, by describing the pathobiological correlates of Powassan genetic diversity, we can better define the potential for tick borne encephalitis to continue to emerge as a public health burden in North America.